Supplementary Materials Supplementary Material supp_142_7_1368__index. tools have already been used to

Supplementary Materials Supplementary Material supp_142_7_1368__index. tools have already been used to study RNA dynamics in cell culture (Armitage, 2011; Santangelo et Perampanel supplier al., 2012); however, they have yet to be widely extended to studies in living vertebrates. One tool, the MS2 RNA-labeling system, based on the high-affinity binding of the bacteriophage MS2 coat protein (MCP) to its RNA hairpin binding site (MBS), has been used in model organisms (Bertrand et al., 1998; Belaya and St Johnston, 2011; Lionnet et al., 2011). By labeling a gene of interest with multiple copies of MBS and coexpressing a fluorescent MCP, live-cell imaging of RNA dynamics with single RNA molecule resolution is possible (Hocine et al., 2013). Tagging the fluorescent MCP with a nuclear localization signal (NLS) primes the MCP for interactions with Perampanel supplier nascent RNAs in the nucleus and reduces cytoplasmic background fluorescence, allowing enhanced visualization of cytoplasmic transcripts. This method has been used to study oogenesis (Belaya and St Johnston, 2011) and embryogenesis (van Gemert et al., 2009), trafficking in oocytes (Gagnon et al., 2013) and recently in brain slices of mice (Park et al., 2014) but has not Mouse monoclonal to Rab25 been applied to vertebrate development. We have developed and applied a Gateway-based MS2-MCP system (Hartley et al., 2000; Walhout et al., 2000; Kwan et al., 2007; Villefranc et al., 2007) for the easy generation of expression vectors and stable transgenic zebrafish lines expressing fluorescent MCPs (FP-MCP). We have validated its use in zebrafish and have used it to study the onset of zygotic transcription and localization in PGCs. Together with established methods of transgenesis and mutagenesis, these tools should facilitate future studies of RNA regulation in living vertebrates. RESULTS AND DISCUSSION Transgenic NLS-tdMCP-GFP lines We developed a set of Gateway-compatible plasmids to facilitate generation of MCP expression vectors (Fig.?1A). Using these plasmids and Tol2-mediated transgenesis (Kawakami et al., 1998, 2004; Kawakami, 2007), we generated stable transgenic zebrafish lines expressing MCP as a tandem dimer (tdMCP) (Wu et al., 2012) fused to a NLS and eGFP under the control of the ubiquitous (Higashijima et al., 1997) and inducible (Halloran et al., 2000) promoters (supplementary material Fig.?S1). In all lines, the labeled cells displayed the expected nuclear fluorescence with minimal cytoplasmic background (supplementary material Fig.?S1). Open in a separate window Fig. 1. Transgenic zebrafish lines expressing NLS-tdMCP-eGFP can be used to detect transcripts (A) Gateway compatible vectors for generation of NLS-tdMCP-FP and MS2-tagged RNAs. Plasmids used to generate transgenic lines by Tol2-mediated transgenesis are shown. 5 Entry plasmids (by recombining the appropriate pME and p3E plasmids. (B) Schematic of the experiment used to validate MS2-MCP interactions. (C-E) Live imaging of embryos at the sphere stage shows that cytoplasmic puncta are visible only in cells expressing the Cherry reporter. In some cases, as shown in C, the Cherry Perampanel supplier reporter aggregates, but does not overlap with MCP-GFP cytoplasmic puncta, suggesting that this is independent of the MS2-MCP interaction. The dotted line denotes borders of cells expressing Cherry reporter. Scale bar: 10?m. Validation of MCP-MBS interaction in zebrafish To validate and test the feasibility of this system to visualize RNA molecules in zebrafish, we transiently and mosaically expressed RNA tagged with MS2 hairpins using the promoter (lines (Fig.?1B). Live imaging of zebrafish embryos revealed tdMCP-GFP cytoplasmic puncta in cells expressing the Cherry protein (Fig.?1C-E), likely representing RNA species, as previously reported in other systems (Bertrand et al., 1998; van Gemert et al., 2009; Lionnet et al., 2011; Schonberger et al., 2012; Gagnon et al., 2013; Park et al., 2014). Furthermore, time-lapse analysis of these cells revealed highly dynamic cytoplasmic puncta (supplementary materials Films?1 and 2) which were not detected in neighboring cells lacking the Cherry reporter (promoter, we co-injected embryos having a Tol2-flanked DNA encoding expressed through the promoter, and RNA encoding the Tol2 Transposase to facilitate genomic integration. Embryos were examined at stages before and after ZGA for nuclear puncta (Fig.?2B). No nuclear puncta were discovered in uninjected embryos anytime stage assayed (supplementary materials Fig.?S2; control plasmid DNA missing.